The present invention is a novel methodology for colormetrically determining the concentration of protein in aqueous media such as certain biological fluids.
The detection of protein in biological fluids such as blood serum and urine is useful in the diagnosis of various abnormalities. For example, in the case of blood serum, both the total protein and the ratio of individual protein fractions may change independently of one another in disease states. Thus, in the case of dehydration, total protein may increase 10 to 15 percent from its normal concentration of approximately 6.8 to 8.8 gm/100 mL, with the increased level being reflected in all protein fractions. Dehydration may result from a decrease in water intake or from excessive water loss as occurs in severe vomiting, diarrhea, Addison's disease and diabetic acidosis. Hypoproteinemia, characterized by total protein levels below 6.0 gm/100 mL is encountered in many unrelated disease states. For example, in the nephrotic syndrome, large masses of albumin may be lost in the urine as a result of leakage of the albumin molecules through the damaged kidney. In salt retention syndromes, water is held back to dilute out the retained salt, resulting in the dilution of all protein fractions. Large quantities of proteins are lost in patients with severe burns, extensive bleeding or open wounds. A long period of low intake or deficient absorption of protein may affect the level and composition of serum proteins, as in sprue and in other forms of intestinal malabsorption as well as in acute protein starvation.
All urines contain some protein. The protein excreted by healthy individuals is on the order of 50 to 100, sometimes as high as 150 mg/24 hour. After considerable muscular exertion, this value may be as high as 250 mg. Proteinuria is said to be present whenever the urinary protein output is greater than that reflected in these normal values. Not all proteinuria is clinically significant, but persistent abnormal levels of protein in the urine is an indicator of the presence of kidney and urinary tract disease. For example, proteinuria may be associated with the early stages of such diseases as pyelonephritis, reflecting bacterial infection in the kidney, and acute glomerulonephritis, often associated with recent streptococcal infections. Proteinuria is associated with certain other disease states which tend to cause kidney lesions such as lupus erythematosus, amyloidosis, toxemia of pregnancy, septicemia and certain forms of drug and chemical poisoning. Thus, the detection of urinary protein and the quantitative assessment of the degree of proteinuria are very important laboratory procedures.
The peroxidase activity of copper protein complexes has been cited in the literature, H. Yamamoto et al Int. J. Biol. Macromol., 2 (4), 263-265, (1980); D. P. Mack et al, J. Am. Chem. Soc., 110, 7572-7574 (1989). Thus, protein bound copper can be used to produce a colored response by catalyzing the oxidation of a redox indicator such as 3,3',5,5'-tetramethylbenzidine by a hydroperoxide. However, it has been discovered that the application of the peroxidase-like activity of the copper protein complex to detect protein in biological fluids, especially serum and urine containing high concentrations of protein, is limited by interferents present in these fluids.
It would be desirable and it is an object of the present invention to provide a method for the determination of proteins in aqueous fluids which involves the peroxidase like activity of protein-copper complexes.
It is a further object to provide such a method in which the interference of components in certain bodily fluids with protein determination is reduced or eliminated.